原发性骨髓纤维化早期骨髓形态学特征研究
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摘要
背景：
BCR-ABL阴性骨髓增殖性肿瘤(myeloproliferative neoplasms, MPN)主要包括真性红细胞增多症(polycythaemia vera, PV)、特发性血小板增多症(essential thrombocythaemia, ET)与原发性骨髓纤维化(primary myelofibrosis, PMF),2008年WHO标准新添JAK2V617F突变作为MPN诊断的一新指标,目前发现约90%的PV患者,50%左右的ET与PMF患者均存在JAK2V617F突变。JAK2V617F突变对BCR-ABL阴性MPN与其他血液病的诊断具有较大价值,但是针对PV、ET与PMF等MPN之间的鉴别诊断,形态学仍是其诊断的基础。尤其是PMF早期与ET,两者的临床表现与实验室检查特征十分相似。PMF早期易转化为纤维化明显期,而真ET具有长期无纤维化进展的特性,因此两者的鉴别诊断对指导临床用药具有重要价值。目前国内关于PMF早期骨髓形态学特征的研究尚未见相关报道,本课题对近八年间156例同步进行骨髓涂片、印片与切片检查的(疑似)ET标本重新进行评估,分析PMF早期与ET的骨髓形态学特征,比较两者的形态学差异。
材料与方法：
骨髓涂片、骨髓印片与骨髓切片标本的采集及染色均由本血液科实验室一步到位。Hemapun959塑料包埋试剂盒购自上海国源生物技术有限公司。JAK2V617突变由武汉海思特康圣达医学检验中心进行检测。
基于PMF早期易进展为纤维化明显期的特性,收集接受一般药物治疗(干扰素或阿司匹林)并且五年内转化为PMF明显期的初诊PMF早期标本7例,分析该7例PMF早期骨髓形态学特点。参照2008年WHO诊断标准,对2003年1月至2011年1月同步进行骨髓涂片、印片与切片检查(疑似)ET的156例标本重新分类,双盲分析PMF早期与ET患者骨髓形态学差异。此外,收集行JAK2V617F突变检测的22例PMF早期与27例ET标本,双盲比较分析JAK2V617F突变型与野生型骨髓的形态学差异。
结果：
五年内转化为纤维化明显期的7例PMF早期骨髓涂片均可见大量血小板生成,可见巨核细胞簇；骨髓印片示7例患者有核细胞量均增多(粒、巨两系为主),易见巨核细胞簇,其中一患者骨髓印片中骨髓基质背景颗粒明显增多；骨髓切片中7例标本有核总细胞量增多、粒细胞量增多、紧密型巨核细胞簇、巨核细胞簇伴异型性改变、小梁旁型巨核细胞、云雾状巨核细胞与小裸核巨均具有较高检出率,3例标本亦检出巨大多分叶巨核细胞。
156例(疑似)ET标本重新分类后示MF-0为34例,MF-1为27例,PMF明显期12例,ET 83例。MF-1组血小板计数、血清乳酸脱氢酶水平显著高于ET组(P<0.05)。骨髓涂片中MF-1组原始细胞比例显著高于ET组(P<0.05)。骨髓切片示MF-0与MF-1组有核细胞量(粒系)增多、紧密型巨核细胞簇、骨小梁旁巨核细胞、云雾状巨核细胞、小裸核巨、大单个圆核巨的检出率均显著高于ET组,而疏松型巨核细胞簇、巨大多分叶核巨核细胞的检出率均显著低于ET组(P均<0.05);MF-1组巨核细胞簇伴异形性改变的检出率显著高于MF-0与ET组(P均<0.05)。PMF早期与ET患者JAK2V617F突变检出率分别为54.5%与48.1%。PMF早期与ET组突变型Hb水平均高于野生型(P均<0.05),而巨核细胞形态在突变型中均未见明显异形性改变。
结论：
1.骨髓切片形态学,尤其是巨核细胞形态学对PMF早期与ET鉴别诊断具有重要价值。
2.骨髓切片形态学参数对PMF早期与ET疾病鉴别诊断的重要性依次为巨核细胞簇伴异形性、云雾状巨核细胞、大单个圆核巨核细胞、有核细胞(粒细胞)总量增多、小裸核巨、骨小梁旁巨核细胞及紧密型巨核细胞簇。
3. JAK2V617F突变对PMF早期及ET患者骨髓巨核细胞形态学无特异影响,但能增强骨髓红系的造血功能。
Background:
BCR-ABL(-) myeloproliferative neoplasms(MPN) mainly included polycythaemia vera(PV), essential thrombocythaemia(ET) and primary myelofibrosis(PMF). In the revised WHO diagnostic standard, JAK2V617F mutaion was added as a reference. It was reported that the JAK2V617F mutaion was detected about in 90% PV,50% ET and 50%PMF. It played a critical role in distinguishing BCR-ABL(-) MPN from other haematological diseases. While to PV, ET and PMF, BM morphology was the still the main way in distinguishing from each other. As to prefibrotic-early PMF and ET, they both have similar clinical presentation, while contrast to ture ET, prefibrotic-early PMF show progression to overt myelofibrosis easily. So it was very important to distinguish from each other. In our study,156 cases labeled as uncertain ET with BM aspirate smears, trephine biopsy sections and imprints conducted simultaneously were recruited, and the BM morphologic character between the prefibrotic-early PMF and ET groups were analyzed.
Materials and methods:
BM aspirate smears, trephine biopsy sections and imprints were taken and processed in our bone marrow (BM) room. JAK2V617F mutation was tested by wu han hai si te kang sheng da company.
As prefibrotic-early PMF show progression to overt myelofibrosis easily, Based on this character,7 cases treated with aspirin or interferon progressed to overt myelofibrosis within five years were selected as prefibrotic-early PMF, the morphology of which was analyzed. Based on the WHO standard,156 cases of uncertain ET with BM aspirate smears, trephine biopsy sections and imprints conducted simultaneously were reclassified, the BM morphologic character between the prefibrotic-early PMF and ET groups were analyzed. In addition,22 cases of prefibrotic-early PMF and 27 ET were selected for morphological comparison between the JAK2V617F mutation positive and negative groups.
Results:
To the seven cases of prefibrotic-early PMF, clusters of megakaryocyte and platelet were found in the BM smear. As to the BM imprint, nucleated cells (granulocytes) were increased in these seven cases, clusters of megakaryocytes were easily found. Granules in the background of BM imprint were obviouly increased in one case. Of the BM section, megakaryocytic cluster with various size, cloud-like megakaryocyte, large ball-like megakaryocyte, increased nucleated cells (granulocyte), small bare nucleus, megakaryocyte near bone trabecula, and compact megakaryocytic cluster could all be found in seven cases, and giant deeply lobulated megakaryocyte was found in three cases.
Of the 156 cases of uncertain ET, it was reclassified as 27 cases of MF-1,12 PMF and 83 ET. The platelet count and LDH level in MF-1 group were obviously higher than ET group (P＜0.05). The blast percentage of BM smear in MF-1 group was also higher than ET group (P<0.05). As to BM section, cases with increased nucleated cells (granulocyte), compact megakaryocytic cluster, megakaryocyte near bone trabecula, cloud-like megakaryocyte, small bare nucleus of megakaryocyte, and large ball-like megakaryocyte in MF-0 and MF-1 group were significantly higher than ET group (all P＜0.05), cases with megakaryocytic cluster of various size in MF-1 group were significantly higher than MF-0 and ET groups(P<0.05). The JAK2V617F mutation rate in prefibrotic-early PMF and ET groups were 54.5% and 48.1%, respectively. Hb level in JAK2V617F mutation positive group was obviously higher than the negative group (P<0.05), no special change with megakaryocytic morphology was found between the positive and negative groups.
Discussion:
1. The morphologic character of BM section, especially megakaryocytic morphology, paly an important role in distinguishing prefibrotic-early PMF from ET.
2. The importance of morphologic index in distinguishing prefibrotic-early PMF from ET were megakaryocytic cluster with various size, cloud-like megakaryocyte, large ball-like megakaryocyte, increased nucleated cells (granulocyte), small bare nucleus, megakaryocyte near bone trabecula and compact megakaryocytic cluster in order.
3. JAK2V617F mutation has no special impact to the megakaryocytic morphology, but it could enhance the hematopoiesis of BM nucleated erythrocytes..
BCR-ABL阴性骨髓增殖性肿瘤(myeloproliferative neoplasms, MPN)主要包括真性红细胞增多症(polycythaemia vera, PV)、特发性血小板增多症(essential thrombocythaemia, ET)与原发性骨髓纤维化(primary myelofibrosis, PMF),2008年WHO标准新添JAK2V617F突变作为MPN诊断的一新指标,目前发现约90%的PV患者,50%左右的ET与PMF患者均存在JAK2V617F突变。JAK2V617F突变对BCR-ABL阴性MPN与其他血液病的诊断具有较大价值,但是针对PV、ET与PMF等MPN之间的鉴别诊断,形态学仍是其诊断的基础。尤其是PMF早期与ET,两者的临床表现与实验室检查特征十分相似。PMF早期易转化为纤维化明显期,而真ET具有长期无纤维化进展的特性,因此两者的鉴别诊断对指导临床用药具有重要价值。目前国内关于PMF早期骨髓形态学特征的研究尚未见相关报道,本课题对近八年间156例同步进行骨髓涂片、印片与切片检查的(疑似)ET标本重新进行评估,分析PMF早期与ET的骨髓形态学特征,比较两者的形态学差异。
材料与方法：
骨髓涂片、骨髓印片与骨髓切片标本的采集及染色均由本血液科实验室一步到位。Hemapun959塑料包埋试剂盒购自上海国源生物技术有限公司。JAK2V617突变由武汉海思特康圣达医学检验中心进行检测。
基于PMF早期易进展为纤维化明显期的特性,收集接受一般药物治疗(干扰素或阿司匹林)并且五年内转化为PMF明显期的初诊PMF早期标本7例,分析该7例PMF早期骨髓形态学特点。参照2008年WHO诊断标准,对2003年1月至2011年1月同步进行骨髓涂片、印片与切片检查(疑似)ET的156例标本重新分类,双盲分析PMF早期与ET患者骨髓形态学差异。此外,收集行JAK2V617F突变检测的22例PMF早期与27例ET标本,双盲比较分析JAK2V617F突变型与野生型骨髓的形态学差异。
结果：
五年内转化为纤维化明显期的7例PMF早期骨髓涂片均可见大量血小板生成,可见巨核细胞簇；骨髓印片示7例患者有核细胞量均增多(粒、巨两系为主),易见巨核细胞簇,其中一患者骨髓印片中骨髓基质背景颗粒明显增多；骨髓切片中7例标本有核总细胞量增多、粒细胞量增多、紧密型巨核细胞簇、巨核细胞簇伴异型性改变、小梁旁型巨核细胞、云雾状巨核细胞与小裸核巨均具有较高检出率,3例标本亦检出巨大多分叶巨核细胞。
156例(疑似)ET标本重新分类后示MF-0为34例,MF-1为27例,PMF明显期12例,ET 83例。MF-1组血小板计数、血清乳酸脱氢酶水平显著高于ET组(P<0.05)。骨髓涂片中MF-1组原始细胞比例显著高于ET组(P<0.05)。骨髓切片示MF-0与MF-1组有核细胞量(粒系)增多、紧密型巨核细胞簇、骨小梁旁巨核细胞、云雾状巨核细胞、小裸核巨、大单个圆核巨的检出率均显著高于ET组,而疏松型巨核细胞簇、巨大多分叶核巨核细胞的检出率均显著低于ET组(P均<0.05);MF-1组巨核细胞簇伴异形性改变的检出率显著高于MF-0与ET组(P均<0.05)。PMF早期与ET患者JAK2V617F突变检出率分别为54.5%与48.1%。PMF早期与ET组突变型Hb水平均高于野生型(P均<0.05),而巨核细胞形态在突变型中均未见明显异形性改变。
结论：
1.骨髓切片形态学,尤其是巨核细胞形态学对PMF早期与ET鉴别诊断具有重要价值。
2.骨髓切片形态学参数对PMF早期与ET疾病鉴别诊断的重要性依次为巨核细胞簇伴异形性、云雾状巨核细胞、大单个圆核巨核细胞、有核细胞(粒细胞)总量增多、小裸核巨、骨小梁旁巨核细胞及紧密型巨核细胞簇。
3. JAK2V617F突变对PMF早期及ET患者骨髓巨核细胞形态学无特异影响,但能增强骨髓红系的造血功能。
Background:
BCR-ABL(-) myeloproliferative neoplasms(MPN) mainly included polycythaemia vera(PV), essential thrombocythaemia(ET) and primary myelofibrosis(PMF). In the revised WHO diagnostic standard, JAK2V617F mutaion was added as a reference. It was reported that the JAK2V617F mutaion was detected about in 90% PV,50% ET and 50%PMF. It played a critical role in distinguishing BCR-ABL(-) MPN from other haematological diseases. While to PV, ET and PMF, BM morphology was the still the main way in distinguishing from each other. As to prefibrotic-early PMF and ET, they both have similar clinical presentation, while contrast to ture ET, prefibrotic-early PMF show progression to overt myelofibrosis easily. So it was very important to distinguish from each other. In our study,156 cases labeled as uncertain ET with BM aspirate smears, trephine biopsy sections and imprints conducted simultaneously were recruited, and the BM morphologic character between the prefibrotic-early PMF and ET groups were analyzed.
Materials and methods:
BM aspirate smears, trephine biopsy sections and imprints were taken and processed in our bone marrow (BM) room. JAK2V617F mutation was tested by wu han hai si te kang sheng da company.
As prefibrotic-early PMF show progression to overt myelofibrosis easily, Based on this character,7 cases treated with aspirin or interferon progressed to overt myelofibrosis within five years were selected as prefibrotic-early PMF, the morphology of which was analyzed. Based on the WHO standard,156 cases of uncertain ET with BM aspirate smears, trephine biopsy sections and imprints conducted simultaneously were reclassified, the BM morphologic character between the prefibrotic-early PMF and ET groups were analyzed. In addition,22 cases of prefibrotic-early PMF and 27 ET were selected for morphological comparison between the JAK2V617F mutation positive and negative groups.
Results:
To the seven cases of prefibrotic-early PMF, clusters of megakaryocyte and platelet were found in the BM smear. As to the BM imprint, nucleated cells (granulocytes) were increased in these seven cases, clusters of megakaryocytes were easily found. Granules in the background of BM imprint were obviouly increased in one case. Of the BM section, megakaryocytic cluster with various size, cloud-like megakaryocyte, large ball-like megakaryocyte, increased nucleated cells (granulocyte), small bare nucleus, megakaryocyte near bone trabecula, and compact megakaryocytic cluster could all be found in seven cases, and giant deeply lobulated megakaryocyte was found in three cases.
Of the 156 cases of uncertain ET, it was reclassified as 27 cases of MF-1,12 PMF and 83 ET. The platelet count and LDH level in MF-1 group were obviously higher than ET group (P＜0.05). The blast percentage of BM smear in MF-1 group was also higher than ET group (P<0.05). As to BM section, cases with increased nucleated cells (granulocyte), compact megakaryocytic cluster, megakaryocyte near bone trabecula, cloud-like megakaryocyte, small bare nucleus of megakaryocyte, and large ball-like megakaryocyte in MF-0 and MF-1 group were significantly higher than ET group (all P＜0.05), cases with megakaryocytic cluster of various size in MF-1 group were significantly higher than MF-0 and ET groups(P<0.05). The JAK2V617F mutation rate in prefibrotic-early PMF and ET groups were 54.5% and 48.1%, respectively. Hb level in JAK2V617F mutation positive group was obviously higher than the negative group (P<0.05), no special change with megakaryocytic morphology was found between the positive and negative groups.
Discussion:
1. The morphologic character of BM section, especially megakaryocytic morphology, paly an important role in distinguishing prefibrotic-early PMF from ET.
2. The importance of morphologic index in distinguishing prefibrotic-early PMF from ET were megakaryocytic cluster with various size, cloud-like megakaryocyte, large ball-like megakaryocyte, increased nucleated cells (granulocyte), small bare nucleus, megakaryocyte near bone trabecula and compact megakaryocytic cluster in order.
3. JAK2V617F mutation has no special impact to the megakaryocytic morphology, but it could enhance the hematopoiesis of BM nucleated erythrocytes..
引文
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[1]卢兴国主编.造血和淋巴组织肿瘤现代诊断学.北京：科学出版社.2005.
[2]卢兴国.检验与临床诊断——骨髓检验分册.北京：人民军医出版社.2007.
[3]Lee SH, Erber WN, Porwit A, et al. ICSH guidelines for the standardization of bone marrow specimens and reports. Int JLab Hematol 2008; 30(5):349-364.
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[6]丛玉隆..医家金鉴.北京：军事医科学出版社：2006.
[7]Pasquale D, Chikkappa G. Comparative evaluation of bone marrow aspirate particle smears, biopsy imprints, and biopsy sections. Am J Hematol 1986; 22(4):381-389.
[8]Sabharwal BD, Malhotra V, Aruna S, et al. Comparative evaluation of bone marrow aspirate particle smears, imprints and biopsy sections. J Postgrad Med 1990; 36(4):194-198.
[9]卢兴国,方淳来,柯莉华,等.骨髓涂片有核细胞量减少的真实性研究.浙江检验医学杂志2006,4(1)：39-41.
[10]范贤斌,朱蕾,卢兴国等.骨髓涂片、印片和切片联检在诊断慢性骨髓增殖性疾病中的意义.实验与检验医学2008,26(3)：281-283.
[11]龚旭波,卢兴国等.骨髓印片在骨髓细胞学检验中的应用价值.中华医学杂志2010；90(22)：1531-1536.
[12]卢兴国等副主编(丛玉隆、李顺义主编).疑难病细胞学诊断.北京：人民卫生出版社.2008：28-73,204-210.
[13]卢兴国.骨髓细胞形态学与病理学.北京：科学出版社.2008：3-13,559-681,1011-1052.
[14]丛玉隆实用检验医学(下).北京：人民卫生出版社.2009：165-192.
[15]Xubo G, Xingguo L, Xianguo W, et al. The role of peripheral blood, bone marrow aspirate and especially bone marrow trephine biopsy in distinguishing atypical chronic myeloid leukemia from chronic granulocytic leukemia and chronic myelomonocytic leukemia. Eur JHaematol 2009; 83(4):292-301.
[16]Lu XG, Huang LS, Xu XH, et al. Application of bone marrow biopsy imprint in evaluating cellularity. Zhejiang Da Xue Xue Bao Yi Xue Ban 2006; 35(3): 331-335.
[17]Gong X, Lu X, Wu X, et al. Role of bone marrow imprints in haematological diagnosis:a detailed study of 3781 cases. Cytopathology 2010 Dec 3. doi: 10.1111/j.1365-2303.2010.00825. x. [Epub ahead of print].
[18]卢兴国,黄连生,许晓华等.骨髓组织印片评估有核细胞量的价值.浙江大学学报(医学版)2006；35(3)：331-335.
[19]张巧红,朱正国,卢兴国.骨髓组织印片在3种恶性肿瘤诊断中的意义探讨.实用医技杂志2005,5(12)：1285-1286.
[20]卢兴国,方淳来,柯莉华,等.骨髓涂片有核细胞量减少的真实性研究.浙江检验医学杂志2006,4(1)：39-41.
[21]吕萍,朱蕾,龚旭波,卢兴国.骨髓组织印片细胞学检查的诊断价值.苏州大学学报(医学版)2008,28(3),426-429.
[22]Orazi A.. Histopathology in the diagnosis and classification of acute myeloid leukemia, myelodysplastic syndromes, and myelodysplastic/myeloproliferative diseases. Pathobiology 2007; 74(2):97-114.
[23]Fend F, Tzankov A, Bink K, et al. Modern techniques for the diagnostic evaluation of the trephine bone marrow biopsy:methodological aspects and applications. Prog Histochem Cytochem 2008; 42(4):203-252.
[24]李顺义.血细胞形态学漏诊分析与对策.中华检验医学杂志2005；28(2)：140-141.
[25]张虹,胡兵.骨髓活检组织印片在贫血诊断中的应用.中国实验血液学杂志 2002；10(2)：131-132.
[26]万楚成,刘瑜,姜铧等.骨髓活检组织印片在急性白血病预后中的临床意义.临床血液学杂志2001；14(6)：264-265.
[27]卢兴国等副主编(丛玉隆主编).医家金鉴.北京：军事医科学出版社.2006：110-211.
[28]Naresh KN, Lampert I, Hasserjian R, et al. Optimal processing of bone marrow trephine biopsy:the Hammersmith Protocol. J Clin Pathol 2006; 59:903-911.
[29]Fend F, Kremer M. Diagnosis and Classification of Malignant Lymphoma and Related Entities in the Bone Marrow Trephine Biopsy. Pathobiology 2007; 74:133-143.
[30]Aboul-Nasr R, Estey EH, Kantarjian HM, et al. Comparison of touch imprints with aspirate smears for evaluating bone marrow specimens.Am J Clin Pathol, 1999; 111(6):753-758.
[31]Kremer M, Quintanilla-Martinez L, Nahrig J, et al. Immunohistochemistry in bone marrow pathology:a useful adjunct for morphologic diagnosis. Virchows Arch,2005; 447(6):920-937.
[32]王鸿利,周新,洪秀华.现代实验诊断学.上海：世界图书出版社,2007：221-234.
[33]Kjurkchiev G, Valkov I. Role of touch imprint and core biopsy for detection of tumor metastases in bone marrow. Diagn Cytopathol 1998; 18(5):323-324.
[34]龚旭波,卢兴国,吴先国,等.CD41标记微小巨核细胞染色法及其临床应用价值探讨.中华血液学杂志2008；29(6)：420-422.
[35]Swerdlow SH, Campo E, Harris NL, et al. World Health Organization Classification of Tumors. Vol 2. Pathology and genetics of tumors of haematopoietic and lymphoid tissues 4th edn. Lyon:IARC Press:2008.